Application of melatonin for the control of food-borne Bacillus species in cherry tomatoes

https://doi.org/10.1016/j.postharvbio.2021.111656Get rights and content

Highlights

  • MLT inhibited the growth of food-borne Bacillus pathogens in cherry tomatoes.

  • MLT repressed cell division and oxidative phosphorylation in B. subtilis.

  • MLT enhanced antioxidant capacity and phenolics biosynthesis in cherry tomatoes.

  • MLT upregulated pathogenesis-related genes in B. subtilis-infected tomatoes.

  • A new application of MLT in postharvest fruit was discovered.

Abstract

Food-borne Bacillus species are often associated with postharvest fruit and vegetables, and are a common cause of food poisoning. In this work, melatonin was found to inhibit the growth of food-borne Bacillus species, including B. cereus, B. licheniformis and B. subtilis, on cherry tomatoes. This result was attributed to two complementary effects. On one side, melatonin showed antibacterial activity to B. subtilis, inhibiting cell division and oxidative phosphorylation, and reducing swimming motility and biofilm formation. On the other side, melatonin enhanced the antioxidant capacity of cherry tomatoes, and induced the biosynthesis of phenolics and ethylene, and the overexpression of pathogenesis-related genes PT16 and PR1b1. The defence response was only observed in the presence of both B. subtilis and melatonin, but not in the single treatments. Although melatonin was known to induce disease resistance in fruit in the presence of necrotrophic pathogens, this is the first report of melatonin inducing fruit defence after treatment with a non-necrotrophic bacterium. Collectively, the application of melatonin for the control of food-borne Bacillus pathogens was explored for the first time, revealing a new potential application of melatonin in postharvest products.

Introduction

Melatonin (MLT, N-acetyl-5-methoxytryptamine) is a multifunctional regulator that is ubiquitously distributed among organisms (Pandi-Perumal et al., 2006; Zhao et al., 2019). MLT was first discovered in the mammalian pineal gland in 1958, and its role in human sleep cycles has been thoroughly explored during last decades (Wang et al., 2020a). In 1995, MLT was identified in plants; since then, MLT has been involved in numerous plant biological processes, including seed germination, growth, flowering, and senescence (Arnao and Hernandez-Ruiz, 2019; Erland et al., 2018; Zhang et al., 2014). MLT concentrations in plants usually range from pico to micrograms per gram of tissue (Wang et al., 2020b); however, its level differs among the species and plant development stages (Di et al., 2019).

MLT is involved in numerous cellular actions as an antioxidant, possessing excellent in vitro and in vivo properties as free radical scavenger, and can protect plants from the damage caused by reactive oxygen species (ROS) (Arnao and Hernandez-Ruiz, 2015). MLT has been demonstrated to enhance plant resistance to pathogen infection (Moustafa-Farag et al., 2020), and this effect has been mainly attributed to the ability of MLT to enhance the antioxidant and photosynthetic capacity on plants (Chen et al., 2018a), and to improve iron homeostasis and secondary metabolism (Ahammed et al., 2020).

Recent studies, mainly reported between 2017 and 2021, have revealed that exogenous MLT is able to promote quality, to enhance tolerance to chilling stress, and to delay senescence in postharvest fruit (Liu et al., 2018; Wang et al., 2020c). The application of MLT in fruit is known to induce the biosynthesis of catalases and peroxidases, reducing the amounts of ROS and malondialdehyde (MDA), and promoting the accumulation of phenolics. Several reports have indicated that MLT is also able to induce innate disease resistance in fruit, and this effect has attributed to MLT-enhanced accumulation of defence hormones salicylic acid, jasmonic acid, nitric oxide and ethylene, and overexpression of defence-related mitogen-activated protein kinases (Wei et al., 2017). This defense reponse is known to induce the overexpression of pathogenesis-related proteins (PRs), and the accumulation of fruit cell wall enzymes chitinase and β-1,3-glucanase (Li et al., 2019). Zhang et al. (2021) reported that the application of MLT in litchi fruit enhanced the activities of phenylalanine ammonia-lyase (PAL), and promoted the accumulation of phenolics and flavonoids, which in turn reduced downy blight symptoms.

Apart from the role of MLT as a plant regulator, MLT has presented effective antibacterial, antifungal and anti-oomycete activities via reduction of amino acid metabolism and virulence (Zhang et al., 2017). MLT showed antibacterial activity against Xanthomonas oryzae pv. oryzae, the causal agent of rice bacterial blight, and at the same time MLT was found to enhance the disease resistance in rice plants against X. oryzae pv. oryzae by inducing the overexpression of PRs PR1b, PR8a and PR9, (Chen et al., 2020, 2018b). Both complementary effects reduced the symptoms produced by the bacterial pathogen in rice plants. In contrast, Li et al. (2019) indicated that MLT did not show any antifungal effect against Botrytis cinerea, but could enhance the disease resistance of tomato fruit against this pathogen by increasing endogenous MLT and salicylic acid contents. Some bioactive natural compounds, such as methyl jasmonate and chitosan, have also been demonstrated to enhance disease resistance, to extend shelf-life of postharvest fruit, and to show antimicrobial activity against postharvest pathogens (Romanazzi et al., 2017; Wang et al., 2021a). In this sense, methyl jasmonate has been thoroughly used for the control of fungal pathogens, mainly Colletotrichum and Penicillium spp. (Tang et al., 2010; Wang et al., 2014).

Postharvest fruit and vegetables can be a suitable platform for the development of microbial ecosystems, which may contain pathogenic microorganisms (van Boekel et al., 2010). The percentage of people suffering from food-borne diseases each year has been reported to be up to 30 %. It is known that more than 250,000 people acquire an infection related to food contaminated by pathogenic microorganisms every year in France, with some of these infections having a fatal outcome (Bridier et al., 2015). Among food-borne pathogens, Bacillus species, including B. cereus, B. licheniformis and B. subtilis, are well known as a cause of food poisoning (Logan, 2012). Although plant pathogens produce necrotic lesions in the fruit, food-borne pathogens are able to form communities on postharvest fruits, but do not produce necrotic lesions. Some food-borne Bacillus species are human pathogens, but not plant pathogens.

In this work, MLT was found for the first time to inhibit the growth of food-borne B. cereus, B. licheniformis and B. subtilis in cherry tomatoes. MLT produced two complementary effects that reduced the presence of B. subtilis in the fruit. B. subtilis was used in the studies due to its easy manipulation, and because it has been thoroughly used as a model organism. On one side, MLT showed antibacterial activity to B. subtilis by inhibiting cell division and oxidative phosphorylation; on the other side, MLT induced an ethylene-based defence response in B. subtilis-infected tomato fruit, and enhanced the biosynthesis of phenolics and flavonoids.

Section snippets

General information and strains

All reagents and chemicals were used as received from commercial suppliers, without further purification or modification. MLT was purchased from Macklin (China). B. cereus BNCC 337166, B. licheniformis BNCC 168439 and B. subtilis 168 were purchased from commercial suppliers and cultured in lysogeny broth (LB; 5 g yeast extract, 10 g tryptone, and 10 g sodium chloride at pH 7.0–7.2 in 1 L of distilled water) at 37 °C and 200 rpm. Cherry tomatoes (Solanum lycopersicum var. cerasiforme) were

MLT inhibits the growth of Bacillus species in cherry tomatoes

MLT reduced the incidence of B. cereus (Fig. 1A), B. licheniformis (Fig. 1B) and B. subtilis (Fig. 1C) in cherry tomatoes. The number of CFU decreased when increasing the concentration of MLT. In this sense, the number of CFU of B. cereus and B. subtilis was reduced by 93 % and 95 %, respectively, after applying 10,000 μM MLT; whereas no B. licheniformis colonies were detected at the same MLT concentration, indicating 100 % inhibition. When studying B. licheniformis and B. subtilis, the

Antibacterial activity of MLT to B. subtilis

Despite the negative effects of food-borne pathogens on human health and their high ability to create resistance to commercial antibiotics, there is a strong lack of methodologies and preservative agents to enhance the safety of postharvest fruit and vegetables (Ahmad et al., 2017).

Some reports have indicated that MLT shows in vitro activity to different bacterial food-borne pathogens, such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii (Atroshi

Conclusion

In summary, MLT was found to reduce the presence of Bacillus species in cherry tomatoes, revealing the potential application of MLT for the control of food-borne pathogens in postharvest fruit. Interestingly, MLT produced 2 complementary effects. On one side, MLT at high concentrations (≥ 1,000 μM) showed strong antibacterial activity against B. subtilis via inhibition of cell division and oxidative phosphorylation. Further, MLT reduced swimming motility and biofilm formation in B. subtilis. On

Funding

This study was supported by the National Natural Science Foundation of China (81803407 and 3201101306), the Nantong Applied Research Program (JC2020103), the Social and Livelihood Project of Nantong (MS12020069), and the Large Instruments Open Foundation of Nantong University (KFJN2130 and KFJN2135).

CRediT authorship contribution statement

Gui-Yang Zhu: Methodology, Investigation, Formal analysis. Peng-Fei Sha: Methodology, Investigation. Xin-Xiao Zhu: Methodology, Investigation. Xin-Chi Shi: Supervision, Resources, Writing - review & editing. Mahdi Shahriar: Supervision, Resources. Yi-Dong Zhou: Supervision. Su-Yan Wang: Conceptualization, Writing - review & editing, Funding acquisition. Pedro Laborda: Project administration, Conceptualization, Writing - original draft.

Declaration of Competing Interest

The authors report no declarations of interest.

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